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Asian Pacific Journal of Tropical Biomedicine logoLink to Asian Pacific Journal of Tropical Biomedicine
. 2011 Jan;1(1):43–48. doi: 10.1016/S2221-1691(11)60066-X

Ovicidal and repellent activities of botanical extracts against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae)

M Govindarajan 1,*, T Mathivanan 1, K Elumalai 2, K Krishnappa 1, A Anandan 1
PMCID: PMC3609154  PMID: 23569723

Abstract

Objective

To determine the ovicidal and repellent activities of methanol leaf extract of Ervatamia coronaria (E. coronaria) and Caesalpinia pulcherrima (C. pulcherrima) against Culex quinquefasciatus (Cx. quinquefasciatus), Aedes aegypti (Ae. aegypti) and Anopheles stephensi (An. stephensi).

Methods

The ovicidal activity was determined against three mosquito species at various concentrations ranging from 50-450 ppm under the laboratory conditions. The hatch rates were assessed 48 h after treatment. The repellent efficacy was determined against three mosquito species at three concentrations viz., 1.0, 2.5 and 5.0 mg/cm2 under the laboratory conditions.

Results

The crude extract of E. coronaria exerted zero hatchability (100% mortality) at 250, 200 and 150 ppm for Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The crude extract of C. pulcherrima exerted zero hatchability (100% mortality) at 375, 300 and 225 ppm for Cx. quinquefasciatus, Ae. aegypti and An. Stephensi, respectively. The methanol extract of E. coronaria found to be more repellenct than C. pulcherrima extract. A higher concentration of 5.0 mg/cm2 provided 100% protection up to 150, 180 and 210 min against Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The results clearly showed that repellent activity was dose dependent.

Conclusions

From the results it can be concluded the crude extracts of E. coronaria and C. pulcherrima are an excellent potential for controlling Cx. quinquefasciatus, Ae. aegypti and An. stephensi mosquitoes.

Keywords: Ervatamia coronaria, Caesalpinia pulcherrima, Ovicidal activity, Repellent activity, Culex quinquefasciatus, Aedes aegypti, Anopheles stephensi, Mosquito, Leaf extract, Mortality

1. Introduction

Mosquito-borne diseases, like malaria, yellow and dengue fevers, are a major threat to over 2 billions people in the tropics[1]. An obvious method for the control of mosquito-borne diseases is the use of insecticides, and many synthetic agents have been developed and employed in the field with considerable success. However, one major drawback with the use of chemical insecticides is that they are non-selective and could be harmful to other organisms in the environment. The toxicity problem, together with the growing incidence of insect resistance, has called attention to the need for novel insecticides[2], and for more detailed studies of naturally-occurring insecticides[3]. Plants may be a source of alternative agents for control of mosquitoes, because they are rich in bioactive chemicals. They are active against a limited number of species including specific target insects, and are bio-degradable. They are potentially suitable for use in integrated pest management programs[4]. The leaf and seed extract of plant Agave americana[5] had mosquito larvicidal properties, and the extract of Tagetes minuta flowers had mosquito larvicidal activity against Aedes aegypti (Ae. aegypti)[6]. The methanolic fraction of leaves of Mentha piperita, Phyllanthus niruri, Leucas aspera and Vitex negundo were against larvae of Culex quinquefasciatus (Cx. quinquefasciatus)[7]. The methanolic extracts of Solanum suratense, Azadirachta indica and Hydrocotyle javanica exhibited larvicidal activity against Cx. quinquefasciatus[8].

More than 1 005 plant species are found to possess insecticidal properties, 384 contain antifeedants, 297 contain repellents, and 27 contain attractants and possess growth inhibitors. All these indicate that the plant kingdom is a vast storehouse of potentially useful chemicals for pest control. It is believed that insect resistance is less likely to occur because many botanicals contain multiactive principles. The pest control principles include properties of insecticide, antifeedant, repellent, chemosterilant, attractant, juvenile and anti-juvenile hormone, moulting and antimoulting hormone, nematicide, rodenticide, fungicide and bactericide[9]–[11]. Sivagnaname and Kalyanasundaram[12] evaluated the methanolic extracts of the leaves of Atlanta monophylla (Rutaceae) for mosquitocidal activity against immature stages of three mosquito species, Cx. quinquefasciatus, Anopheles stephensi (An. stephensi) and Ae. aegypti in the laboratory. Vasudevan et al[13] noted that ovicidal activity of castor oil extracted from castor seeds against mosquitoes An. stephensi, Culex fatigans and Ae. aegypti. Prakash[14] stated that ovicidal action of certain chitin synthesis inhibitors diflubenzuron, penfluron and bay SIR 8514 against mosquitoes, Cx. quinquefasciatus, Ae. aegypti, An. stephensi and Anopheles culicifacies (An. culicifacies). Ovicidal effect of the seed extract of Atriplex canescens was reported against Cx. quinquefasciatus[15]. Su and Mulla[16] reported that the ovicidal activity of neem products Azadirachtin against mosquitoes Culex tarsalis (Cx. tarsalis) and Cx. quinquefasciatus. Rajkumar and Jebanesan[17] studied that ovicidal activity of Solanum trilobatum leaf extract against Cx. quinquefasciatus and Cx. tritaeniorhynchus.

Larvicidal efficacy of the crude leaf extract of Ficus benghalensis with three different solvents like methanol, benzene and acetone was tested against the early second, third, fourth instar larvae of Cx. quinquefasciatus, Ae. aegypti and An. Stephensi[18]. The acetone, chloroform, ethyl acetate, hexane and methanol leaf extracts of Acalypha indica, Achyranthes aspera, Leucas aspera, Morinda tinctoria and Ocimum sanctum were studied against the early fourth-instar larvae of Ae. aegypti L and Cx. quinquefasciatus Say[19]. Larvicidal activities of crude hexane, ethyl acetate, petroleum ether, acetone, and methanol extracts of the leaf of five species of cucurbitaceous plants, Citrullus colocynthis, Coccinia indica, Cucumis sativus, Momordica charantia, and Trichosanthes anguina, were tested against the early fourth instar larvae of Ae. aegypti L. and Cx. quinquefasciatus[20]. The benzene and methanol extracts of Artemisia vulgaris have repellent activity against Ae. aegypti[21]. The Zanthoxylum armatus, Zanthoxylum alatum (Rutaceae), Azadirachta indica (Mailiaceae) and Curcuma aromatica (Zingiberaceae) possess repellent properties against mosquitoes[22]. The repellent activity of active compound octacosane was from Moschosma polystachyum against the vector Cx. quinquefasciatus[23]. The essential oil of Zingiber officinalis was used as a mosquito larvicidal and repellent agent against the filarial vector Cx. quinquefasciatus[24]. The present study was carried out to determine the ovicidal and repellent efficacy of Ervatamia coronaria (E. coronaria) and Caesalpinia pulcherrima (C. pulcherrima) plant leaves extract against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.

2. Materials and methods

2.1. Plant collection

Fully developed leaves of the E. coronaria and C. pulcherrima were collected from in and around Annamalai University Campus, Annamalainagar, Tamil Nadu, India. It was authenticated by a plant taxonomist from the Department of Botany, Annamalai University. A voucher specimen was deposited at the Herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University.

2.2. Extraction

The dried leaves (1.0 kg) were extracted with methanol (3.0 L) in a soxhlet apparatus and the extract was evaporated in a rotary vacuum evaporator to yield a dark greenish mass. Standard stock solutions were prepared at 1% by dissolving the residues in methanol, which was used for the bioassays.

2.3. Mosquitoes

The mosquitoes, Cx. quinquefasciatus, Ae. aegypti and An. stephensi were reared in the Vector Control Laboratory, Department of Zoology, Annamalai University. The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio. Adults were provided with 10% sucrose solution and one week old chick for blood meal. Mosquitoes were held at (28±2)°C, 70%-85% relative humidity (RH), with a photo period of 14 h light, 10 h dark.

2.4. Ovicidal activity

Ovicidal activity was assessed by the slightly modified method of Su and Mulla[16]. The egg raft/eggs of Cx. quinquefasciatus, An. stephensi and Ae. aegypti were collected from Vector Control Laboratory, Annamalai University. The different leaf extract was diluted in the appropriate solvent to achieve various concentrations ranging from 50 to 450 ppm. Eggs of these mosquito species (100 nos.) were exposed to each concentrations of leaf extract. After treatment the eggs from each concentration were individually transferred to distilled water cups for hatching assessment after counting the eggs under microscope. Each experiment was replicated six times along with appropriate control. The hatch rates were assessed 48 h after treatment by the following formula.

2.4.

2.5. Repellent activity

The repellent study was following the method of WHO[25]. Three-day-old blood-starved female Cx. quinquefasciatus and An. stephensi mosquitoes (100) were kept in a net cage (45 cm × 30 cm × 45 cm). The volunteer had no contact with lotions, perfumes or perfumed soaps on the day of the assay. The arms of volunteer, only 25 cm2 dorsal side of the skin on each arms was exposed and the remaining area covered by rubber gloves. The crude extract was applied at 1.0, 2.5 and 5.0 mg/cm2, separately in the exposed area of the forearm. Only ethanol served as control. The time of the test depended on whether the target mosquitoes day-or night biters. Ae. aegypti was tested during the day time from 7:00 to 17:00, while Cx. quinquefasciatus and An. stephensi were tested during the night from 19:00 to 5:00. The control and treated arms were introduced simultaneously into the mosquito cage, and gently tapping the sides on the experimental cages, the mosquitoes were activated. Each test concentration was repeated six times. The volunteer conducted their test of each concentration by inserting the treated and control arms into the same cage for one full minute for every five minutes. The mosquitoes that landed on the hand were recorded and then shaken off before imbibing any blood; making out a 5 min protection. The percentage of repellency was calculated by the following formula.

% Repellency= [(Ta – Tb)/Ta] ×100

Where Ta is the number of mosquitoes in the control group and Tb is the number of mosquitoes in the treated group.

2.6. Statistical analysis

The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 and other statistics at 95% confidence limits of upper confidence limit and lower confidence limit, and Chi-square values were calculated using the SPSS12.0 (Statistical Package of Social Sciences) software. Results with P<0.05 were considered to be statistically significant.

3. Results

The methanol extracts of E. coronaria and C. pulcherrima have been studied for use as natural insecticides instead of organic phosphorous materials or other synthetic agents. Results on the ovicidal and repellent effects of leaf extract were reported in the present study and confirmed their potential for control of the mosquito populations (Table 1–4). The crude extract of E. coronaria exerted zero hatchability (100% mortality) at 250, 200 and 150 ppm for Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The crude extract of C. pulcherrima exerted zero hatchability (100% mortality) at 375, 300 and 225 ppm for Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The methanol extract of E. coronaria was found to be more repellent than C. pulcherrima extract. A higher concentration of 5.0 mg/cm2 provided 100% protection up to 150, 180 and 210 min against Cx. quinquefasciatus, Ae. aegypti and An. stephensi, respectively. The repellent activity was very high at the initial stage of exposure. Increase in the exposure period showed reduction in repellent activity and it depends upon the concentration of the extract and density of mosquito. The results clearly showed that repellent activity was dose dependent.

Table 1. Ovicidal activity of methanol extract of E. coronaria against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.

Mosquito Percentage of egg hatch ability
Control 50 ppm 100 ppm 150 ppm 200 ppm 250 ppm 300 ppm
Cx. quinquefasciatus 100.0±0.0 66.8±1.9 45.6±1.7 31.2±1.9 23.8±1.3 NH NH
Ae. aegypti 100.0±0.0 57.9±1.5 38.2±0.8 26.4±1.9 NH NH NH
An. stephensi 100.0±0.0 51.2±1.8 34.6±1.5 NH NH NH NH
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Each value (χ±SD) represents the mean of six values; NH: No hatchability (100% mortality).

Table 4. Repellent activity of crude methanol extract of C. pulcherrima against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.

Mosquitoes Concentration (mg/cm2) % of repellency
15 min 30 min 60 min 90 min 120 min 150 min 180 min 210 min
Cx. quinquefasciatus 1.0 100.0±0.0 100.0±0.0 100.0±0.0 78.2±0.8 68.8±1.5 53.1±1.8 36.5±1.2 31.2±0.9
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 76.3±1.2 66.4±1.5 47.4±1.5 38.9±1.8
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 78.9±1.3 69.9±1.4 58.4±1.9
Ae. aegypti 1.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 80.8±1.8 63.7±1.6 61.7±1.6 56.6±1.6
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 81.6±1.4 72.3±1.2 66.0±1.8
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 83.5±2.1 73.5±1.3
An. stephensi 1.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 79.3±1.5 68.4±1.6 59.0±1.4
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 83.3±1.5 75.5±1.8
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 82.1±2.2
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Each value (χ±SD) represents average of six values.

Table 2. Ovicidal activity of methanol extract of C. pulcherrima against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.

Mosquito Percentage of egg hatch ability
Control 75 ppm 150 ppm 225 ppm 300 ppm 375 ppm 450 ppm
Cx. quinquefasciatus 100.0±0.0 66.4±1.5 49.8±1.2 32.6±1.6 23.4±1.2 NH NH
Ae. aegypti 100.0±0.0 59.6±1.2 37.4±1.6 23.8±1.7 NH NH NH
An. stephensi 100.0±.0.0 54.6±1.5 28.8±1.6 NH NH NH NH
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Each value (χ±SD) represents the average of six values; NH: No hatchability (100% mortality).

Table 3. Repellent activity of crude methanol extract of E. coronaria against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.

Mosquitoes Concentration (mg/cm2) % of repellency
30 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min
Cx. quinquefasciatus 1.0 100.0±0.0 100.0±0.0 100.0±0.0 83.2±1.2 72.6±1.9 64.3±2.2 56.5±1.6 41.6±1.6
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 84.3±0.0 79.2±1.8 68.2±1.4 59.4±1.2
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 88.7±1.2 88.9±1.9 74.3±1.8
Ae. aegypti 1.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 79.2±1.8 71.3±0.8 61.7±1.7 52.0±1.9
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 82.2±1.7 73.5±1.2 64.2±1.3
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 89.7±0.8 76.8±1.4
An. stephensi 1.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 78.0±1.5 69.2±1.9 57.7±1.2
2.5 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 86.4±1.4 78.2±0.8
5.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 87.4±1.4
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Each value (χ ± SD) represents average of six values.

4. Discussion

The results of entomotoxicity of E. coronaria and C. pulcherrima crude leaf extract were also comparable with earlier reports. Larvicidal and ovicidal activity of crude hexane, ethyl acetate, benzene, chloroform and methanol extracts of the leaf of three plants, Eclipta alba (E. alba), Cardiospermum halicacabum (C. halcacabum), and Andrographis paniculata (A. paniculata), were tested against the early third instar larvae of An. stephensi, the highest larval mortality was found in methanol extract of A. paniculata, E. alba and C. halicacabum against the larvae of An. stephensi (LC50=79.68, 112.56 and 133.01 ppm; LC90=154.66, 220.68 and 270.72 ppm), respectively. The percent hatchability was inversely proportional to the concentration of extract. Mortality of 100% with methanol and ethyl acetate extract of A. paniculata and methanol extract of E. alba was exerted at 200 ppm and methanol and benzene extract of C. halicacabum exerted at 150 ppm[26]. Methanolic leaf extract of Cassia fistula was tested for larvicidal activity against Cx. quinquefasciatus and An. stephensi[27]. The leaf extract of Acalypha indica with different solvents viz, benzene, chloroform, ethyl acetate and methanol was tested for larvicidal, ovicidal activity and oviposition attractancy against An. stephensi. The larval mortality was observed after 24 h exposure. The LC50 values were 19.25, 27.76, 23.26 and 15.03 ppm, respectively[28]. The extract was found to be more lethal to the larvae of An. stephensi than Cx. quinquefasciatus with LC50 values of 17.97 and 20.57 mg/L, respectively. The extracts of Quercus iusitania var. infectoria galls (Oliv.) showed larvicidal activity and their possible use in biological control of Culex pipines[29]. The LC50 values are 335 and 373 ppm for the 2nd and 4th instar larvae. The leaf extract of Cassia fistula with different solvents viz, methanol, benzene and acetone was studied for the larvicidal, ovicidal and repellent activity against Ae. aegypti. The 24 h LC50 concentration of the extract against Ae. aegypti were observed at 10.69, 18.27 and 23.95 mg/L, respectively[30]. The mosquito larvicidal properties of the leaf extract of a herbaceous plant, Ocimum canum against Ae. aegypti was observed. The LC50 values for 2nd, 3rd and 4th instar larvae were 177.82, 229.08 and 331.13 ppm, respectively[31]. Phytochemicals obtained from huge diversity of plant species are an important source of safe and biodegradable chemicals which could be screened for mosquito repellent and insecticidal activities. Repellents of plant origin do not pose hazards of toxicity to human and domestic animal and are easily biodegradable. Natural products are safe for human when compared with synthetic compounds[32].

Rahuman and Venkatesan[33] reported the petroleum ether extract of Citrullus colocynthis, methanol extracts of Cannabis indica, Cannabis sativus, Momordica charantia and acetone extract of Trichosanthes anguina against the larvae of Ae. aegypti (LC50=74.57, 309.46, 492.73, 199.14, and 554.20 ppm) and against Cx. quinquefasciatus (LC50=88.24, 377.69, 623.80, 207.61 and 842.34 ppm), respectively.Larvicidal efficacies of methanol extracts of Momordica charantia, Trichosanthes anguina, Luffa acutangula, Benincasa cerifera and Citrullus vulgaris tested with LC50 values were 465.85, 567.81, 839.81, 1 189.30 and 1 636.04 ppm, respectively, against the late third larval age group of Cx. quinquefasciatus[34]. The ethanol extracts of the aerial parts from five Labiatae species, Teucrium divaricatum was the most toxic, followed by Mentha longifolia, Melissa officinalis, Salvia sclarea and Mentha pulegium against the third- and fourth-instar larvae of Culex pipiens with LC50 values of 18.6, 26.8, 39.1, 62.7 and 81.0 ppm, respectively[35]. Extract from Lavandula stoechas (Labiatae) showed LC50 values of 89 mg/L against fourth instar larvae of Culex pipiens molestus[36]. Mean median lethal concentration values of the aqueous extract from the roots of Hibiscus abelmoschus (Malvaceae) against the larvae of An. culicifacies, An. stephensi and Cx. quinquefasciatus were 52.3, 52.6 and 43.8 ppm, respectively[37]. Larvicidal efficacy of leaf extracts of Pavonia zeylanica and Acacia ferruginea (Malvaceae) were tested against the late third-instar larvae of Cx. quinquefasciatus, and their LC50 values were 2 214.7 and 5 362.6 ppm, respectively[38].

Mullai and Jebanesan[39] have reported that ethyl acetate, petroleum ether and methanol leaf extracts of Citrullus colocynthis and Cucurbita maxima showed LC50 values of 47.58, 66.92 and 118.74 ppm and 75.91, 117.73 and 171.64 ppm, respectively, against Cx. quinquefasciatus larvae. Rahuman et al[40] have reported that the LC50 value of petroleum ether extracts of Jatropha curcas, Pedilanthus tithymaloides, Phyllanthus amarus, Euphorbia hirta, and Euphorbia tirucalli were 8.79, 55.26, 90.92, 272.36 and 4.25 ppm, respectively, against Ae. aegypti and 11.34, 76.61, 113.40, 424.94 and 5.52 ppm, respectively, against Cx. quinquefasciatus. Karunamoorthi et al[41] reported that the petroleum ether (60-80 °C) extracts of the leaves of V. negundo were evaluated for larvicidal activity against larval stages of Cx. tritaeniorhynchus in the laboratory with LC50 and LC90 values of 2.488 3 and 5.188 3 mg/L, respectively. The methanol leaf extracts of V. negundo, Vitex trifolia (V. trifolia), Vitex peduncularis (V. peduncularis) and Vitex altissima (V. altissima) possessed varying levels of larvicidal activity on Cx. quinquefasciatus and An. stephensi and found with LC50 value of 212.57, 41.41, 76.28 and 128.04 ppm, respectively[42]. The peel methanol extract of Citrus sinensis and the leaf and flower ethyl acetate extracts of Ocimum canum were tested against the larvae of An. stephensi (LC50=95.74, 101.53, 28.96, LC90=303.20, 492.43 and 168.05 ppm), respectively[43]. The larvicidal effect of ten plants corresponds to different botanical families on An. stephensi and Cx. quinquefasciatus. The highest larval mortality was found in leaf acetone and methanol of Canna indica (LC50= 29.62 and 40.77 ppm; LC90=148.55 and 165.00 ppm) against second instar larvae (LC50=121.88 and 69.76 ppm; LC90=624.35 and 304.27 ppm) and against fourth instar larvae of methanol and petroleum ether extracts of Ipomoea carnea (LC50= 41.82 and 39.32 ppm; LC90=423.76 and 176.39 ppm) against second instar larvae (LC50=163.81 and 41.75 ppm; LC90= 627.38 and 162.63 ppm) and against fourth instar larvae of Cx. quinquefasciatus, respectively[44]. This study reveals that the E. coronaria and C. pulcherrima has remarkable ovicidal and repellent properties against Cx. quinquefasciatus, Ae. aegypti and An. stephensi mosquitoes. The flora of India has rich aromatic plant diversity with potential for development of natural insecticides for control of mosquito and other pests. These results could encourage the search for new active natural compounds offering an alternative to synthetic repellents and insecticides from other medicinal plants.

Acknowledgments

The Authors are grateful to the Dr. (Mrs) Selvi Sabhanayakam, Professor and Head, Department of Zoology, Annamalai University for the laboratory facilities provided. We acknowledge the staff members of the VCRC (ICMR), Pondicherry for their cooperation.

Footnotes

Conflict of interest statement: We declare that we have no conflict of interest.

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